Method for the immunization of animals and men against Toxoplasma gondii _

ABSTRACT

A method for the immunization of mammals (including man) and birds, against Toxoplasma gondii is described which involves subcutaneous administration of a live, low-virulent vaccine comprising a temperature-sensitive mutant of the persistent, virulent RH strain of T. gondii. The vaccine gives immunity against subsequent challenge, but does not persist and does not cause chronic infection of the subject and which therefore cannot result in illness or death in the event that the subject later becomes immunosuppressed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with a method for immunizing animalsof various types against Toxoplasma gondii. More particularly, it isconcerned with such a method which makes use of a specific mutant of T.gondii which has surprisingly been found to give immunity withoutconcomitant chronic infection of the animal.

2. Description of the Prior Art

Toxoplasmosis is an animal disease that can be transmitted to man,typically through contact with cat feces or raw meat. The disease iscommon to all domestic animals, including barnyard species. Ultimately,however, the possibility of human infection presents the most seriousproblem.

The spectrum of human disease due to Toxoplasma was characterized by acombination of serologic, immunologic and epidemological studies, and byisolation of Toxoplasma. In the acute infection where cells aredestroyed by rapidly proliferating organisms, there may occur fever,pneumonia, an inflammation of the heart muscle, liver and skin (rash).Toward the end of this period or following a subclinical acuteinfection, localized or generalized swelling of lymph nodes is observed,especially in women. In newborn infants infected in utero, a subacutedisease picture is typical. This results from the attenuation of thedisease process by the passive immunization conferred by the mother, andthe slow acquisition of active immunity due to immaturiy of the fetusand the newborn. In addition to the symptoms of acute Toxoplasmosismentioned above, meningoencephalitis ("brain fever"), often withhydrocephalus ("water on the brain"), and retinochoroiditis (intraocularinflammation) are important. Most of the mothers who have given birth toinfected babies had asymptomatic infections.

Thus, Toxoplasmosis deserves special attention because of the seriousdanger it raises for the unborn human baby. A pregnant woman may havethe infection and unknowingly infect the fetus. Even if diagnosed andtreated, the child may be born with permanent brain and eye damage.Diagnosis during pregnancy is uncertain at best and treatment uncertainand risky. For this reason, efforts to prevent infection duringpregnancy are important.

It has been known in the past that mammals can be immunized againstToxoplasmosis. The procedure is very typical, and involvesadministration of a strain of the organism so that the mammal can buildup immunity accompanied by specific antibodies against subsequent T.gondii challenge. This straightforward procedure presents a number ofproblems. For example, primary infection of cats (an important carrierof Toxoplasma) is usually followed by oocyst shedding before buildup ofimmunity. This phenomenon largely defeats the purpose of immunization,in that infective oocysts in the cat feces are a prime carrier of thedisease. Furthermore, all known strains of the organism used for primaryinfections of mammals, while effective for purposes of building upimmunity, tend to persist in the mammal for a long period of time, andpossibly for a lifetime, with the result that the mammal is chronicallyinfected. This in turn raises the possibility that if such a mammalbecomes immunosuppressed later in life, the infection may reactivatewith debilitating or even fatal results.

There is therefore a decided and heretofore unsatisfied need for amethod of immunizing animals against T. gondii which on the one handgives an adequate level of immunity, but on the other does not persistand cause chronic infection in the animal.

SUMMARY OF THE INVENTION

The present invention overcomes the problems noted above, and provides amethod of immunizing animals selected from the group consisting of man,cattle, sheep, goats, birds and zoo mammals (e.g., members of the catfamily, new-world monkeys and kangaroos) against Toxoplasma gondii. Themethod involves subcutaneously administering to the animal an amount ofa low-virulent mutant of T. gondii discovered to have the ability todevelop in the animal an immunity to subsequent challenge. Moreover,this immunity is developed without concomitant chronic T. gondiiinfection in the animal.

The mutant particularly found useful in connection with the invention isdescribed in "Toxoplasma gondii: Isolation and PreliminaryCharacterization of Temperature-sensitive Mutants" by E. R. Pfefferkornet al., Experimental Parasitology, Volume 39, pages 365-376 (1976), asthe "ts-4" mutant. The above paper is hereby expressly incorporated byreference herein. In addition, a publicly available frozen inoculum ofthe "ts-4" mutant described in the referenced paper has been depositedwith The American Type Culture Collection, Rockville, Md. This culturehas been designated "ts-4", and has an accession number of 40050.

The T. gondii mutant in accordance with the invention is usuallysubcutaneously administered in the form of a vaccine, using a salinesolution as carrier. Inasmuch as the mutant is injected live, the amountinjected is not critical; however, it is believed that a level of atleast about 20 tachyzoites of the mutant should be injected. In certaincases multiple vaccinations have proved helpful in assuring completeimmunity.

DESCRIPTION OF THE PREFERRD EMBODIMENTS Production and Selection ofToxoplasma gondii Mutant 1. Materials and Methods

All tissue culture experiments were done with human diploid foreskinfibroblasts in their 15th to 35th passage. These cells were grown inEagle's (Eagle, H., "Nutritional needs of mammalian cells in tissueculture", Science 122, pp. 501-504 (1955)) minimal essential mediumcontaining Earle's (Earle, W. R., "Production of malignancy in vitro.IV. The mouse fibroblast cultures and changes seen in the living cells",Journal of the National Cancer Institute 4, pp. 165-212 (1943) salts andsupplemented with 10% inactivated (56° C. for 30 min) fetal calf serum,100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/mlamphotericin B. The serum used was negative for antibody to T. gondii inthe Sabin-Feldman (Sabin, A. B., and Feldman, H. A., "Dyes asmicrochemical indicators of a new immunity phenomenon affecting aprotozoan parasite (Toxoplasma)." Science 108, pp. 660-663 (1948)) dyetest performed as described by Frenkel and Jacobs (Frenkel, J. K. andJacobs, I., "Ocular toxoplasmosis", Archives of Ophthalmology 59, pp.260-279 (1958)). The cultures were incubated under 5% CO₂ in plastictissue culture flasks or in disposable glass roller bottles. Theirmedium was replaced twice weekly and the cultures were split 1:3 whenconfluent. All infections with T. gondii were carried out in the mediumdescribed above with the concentration of inactivated fetal calf serumreduced to 3%. At this serum concentration, confluent monolayer culturesmaintained their normal morphology for at least two weeks.

Toxoplasma gondii, strain RH, was in the form of a mouse peritonealexudate. This strain was cloned twice successively. The final productwas designated as the wild type. This wild-type T. gondii was maintainedin the laboratory by passage in tissue culture at 37° C. Massivelyinfected monolayers were dispersed into their medium by mechanicalscraping, and the resulting suspension of extracellular andintracellular parasites was diluted 5- to 300-fold into fresh cultures.Temperature-sensitive mutants of T. gondii were maintained by similartissue culture passage at 33° C.

Both mutant and wild-type parasites were preserved by storage in sealedglass ampoules under liquid nitrogen. (Bollinger, R. O., Musallam, N.and Stulberg, C. S., "Freeze preservation of tissue culture propagatedToxoplasma gondii," Journal of Parasitology 60, pp. 368-369 (1974)). Themedium used for freezing contained Eagle's (1955) minimal essentialmedium in Hanks' salts (Hanks, J. H., and Wallace, R. E., "Relation ofoxygen and temperature in the preservation of tissues by refrigeration",Proceedings of the Society for Experimental Biology and Medicine, 71,pp. 196-200 (1949)) supplemented with 10% inactivated fetal calf serum,5% glycerol, and 5% dimethylsulfoxide. The recovery of viable parasiteswas significantly improved by slowly reducing the concentration of thesecryoprotective agents over a period of 30 min. after thawing.

Before massive infection and host cell lysis supervene, most of theparasites in an infected culture are intracellular. Accurate plaquetitrations and the isolation of mutants require the use of extracellularparasites. These were prepared in one of two ways, either one of whichyielded suspensions in which microscopic examination showed that morethan 95% of the parasites were extracellular. Plaque assays of variouspreparations showed that 12 to 75% of the wild-type extracellularparasites were infectious. The comparable values for temperaturesensitive mutants were 6 to 50%. In the first method, the medium of aninfected monolayer was decanted and the cells were scraped from theplastic surface with a rubber-tipped glass rod. Vigorous pipettingserved to disperse the cells in 1 ml of fresh medium that was thentransferred to a 4 ml vial. The cells in this suspension were disruptedby three forced passages through a 27-gauge needle attached to a 5 mlsyringe (Chaparas, S. D., and Schlesinger, R. W., "Plaque assay ofToxoplasma on monolayers of chick embryo fibroblasts", Proceedings ofthe Society for Experimental Biology and Medicine, 102, pp. 431-437(1959)). In the second method, the medium of an infected culture wasdecanted and replaced with an equal volume of fresh medium. After 1/2 to2 hr. incubation, the paraistes newly released by spontaneous lysis werecollected by again decanting the medium. Nearly all of the infected anduninfected cells remained attached to the plastic culture vessel.

Published methods for the plaque titration of T. gondii under an agaroverlay (Chaparas and Schlesinger (1959); Akinshina, G. T., andZasukhina, G. D., Method of investigating mutations in protozoa(Toxoplasma gondii), Genetika 2, pp. 71-75, (1966); and Foley, V. L. andRemington, J. S., "Plaquing of Toxoplasma gondii in secondary culturesof chick embryo fibroblasts", Journal of Bacteriology 98, pp. 1-3(1969)) were tested and it was concluded that a procedure using a liquidoverlay was simpler and reliable. Human diploid fibroblast cells weregrown to confluency in 25 cm² plastic tissue culture flasks. The mediumwas discarded and replaced with fresh medium containing 3% inactivatedfetal calf serum. Suspensions of extracellular T. gondii were diluted inEagle's (1955) minimal essential medium in Hanks' (Hanks and Wallace1949) salts and 3% serum and small volumes of suitable dilutions wereadded to the flasks. The flasks were then incubated undisturbed for 4 to7 days, at 37° or 40° C., or 9 to 14 days at 33° C. The infectiousparasites produced irregularly shaped plaques that were 1-2 mm indiameter. The irregular shape is a consequence of the oriented growth ofour human fibroblasts. Macroscopically visible secondary plaques werenot observed unless the culture was disturbed early in the incubationperiod. The plaques were counted without the aid of staining through theuse of oblique illumination and a dark background. Preliminaryexperiments showed that staining the infected monolayers with cyrstalviolet did not reveal additional plaques that were uncounted inunstained preparations. The data from our titrations are recorded asplaque forming units (PFU).

Selection of mutants requires an efficient method of cloning. LinbroDisposo-Trays were employed that contain 96 flat-bottomed wells 7 mm indiameter. These wells were seeded with 0.1 ml of medium containing about10⁵ cells and a small number of parasites, estimated to lie between 0.3and 3 PFU per well. This estimation was based on a microscopicenumeration of the extracellular parasites corrected for the probabledegree of infectivity. The microwell cultures were incubated in a glassjar that was gassed with 5% CO₂ and then sealed. After an appropriateinterval, 1 week for incubation at 37° and 40° C. or 2 weeks at 33° C.,each individual culture was examined with an inverted microscope and thelocation of wells containing but a single plaque was noted. Theparasites in these wells were assumed to be a clone. This conclusion islikely to be valid, for the distribution of plaques among the wellscorresponded closely to the expected Poisson distribution. Both thewild-type and the mutant strains were cloned twice to give addedassurance of genetic purity.

To permit chemical mutagenesis, extracellular parasites were allowed toinfect about 10% of the cells of a confluent monolayer. At the time ofinfection N-methyl-N'-nitro-N-nitrosoguanidine (Aldrich ChemicalCompany) was added to the medium at a concentration of 0.8 μg/ml. After24 hr incubation at 33° C., the mutagen was removed by three rinses withmedium and the cultures were incubated in fresh medium at 33° C. for twoadditional days. Extracellular parasites were then prepared from theresulting massively infected culture and used in the isolation oftemperature-sensitive mutants.

The rate of growth of T. gondii in tissue culture was measured in twoways. In the first, intracellular parasites were released by passing acell suspension through a 27 gauge needle as described above. Theresulting extracellular parasites were pooled with those previouslyspontaneously released into the medium and the total infectivity wasdetermined by a plaque titration. This procedure was repeated at regularintervals using replicate cultures, and the data from the resultingtitrations were used to construct a growth curve. Since this method wascumbersome when the rates of growth of several mutants weresimultaneously to be determined at different temperatures, we also useda procedure that depended on microscopic enumeration. Replicate cultureswere infected with about 10¹ PFU and incubated undisturbed. Atintervals, the total number of parasites in the resulting microfoci ofinfection was counted. This enumeration was done using unstained cellsunder 300-fold phase-contrast magnification. The individual parasites ofat least 50 foci per flask were counted when the average per focus wasless than 80. At higher values, the parasites of only 25 foci werecounted. At fewer than 200 parasites per focus, the results were highlyreproducible and yielded exponential growth curves, except whentemperature-sensitive mutants were studied at temperatures that wereinhibitory to growth. When these two methods were used in replicateinfected cultures they yielded exponential growth curves of the sameslope.

2. Results Permissive and Restrictive Temperatures

The first step in the isolation of temperature-sensitive mutants issetting the permissive and restrictive temperatures. To this end, theability of the wild-type T. gondii to grow in a plaque assay at varioustemperatures was successful. Plaques were produced over the range of 30°to 41° C. Growth at such a high temperature has also been noted byAkinshina and Zasukhina (1966). 40° C. was chosen as the restrictivetemperature to be sure that minor fluctuations of incubator temperaturewould not affect the growth of the wild-type parasite. For thepermissive temperature 33° C. was chosen; a compromise between a desireto have as great a span as possible between the two temperatures and adesire for a relatively rapid plaque assay. The efficiency of plating,the ability of the wild-type T. gondii to make plaques, was notcompromised at either extreme of temperature. Table 1 compares the PFUtiter of a single preparation of extracellular wild-type T. gondii understandard conditions, 37° C., and at the permissive and restrictivetemperatures that were established. The slower growth of T. gondii at33° C. required a longer incubation for the assay but the resultingplaques, as shown in FIG. 1, were roughly the same size. Furthermore,the data of Table I shows that temperature had no significant effect onthe plating efficiency of the wild-type T. gondii.

                  TABLE I                                                         ______________________________________                                        Efficiency of Plating of Wild Type                                            Toxoplasma gondii at Different Temperatures                                   Temperature  Plaques counted in                                               (C)          quadruplicate flasks*                                                                        Average                                           ______________________________________                                        40           432, 423, 408, 396                                                                           415                                               37           404, 394, 423, 432                                                                           413                                               33           430, 429, 429, 412                                                                           425                                               ______________________________________                                         *These plaques were enumerated early in the incubation period (4 days at      37° and 40° C. or 9 days at 33° C.) to allow the         reliable counting of large numbers.                                      

Mutagenesis

Treatment with chemical or physical mutagenic agents is generally anessential step in the isolation of mutants of eukaryotic cells. This isparticularly true in the search for temperature-sensitive mutants whichmay have no selective advantage that can be exploited to eliminate theundesired wild-type organisms. Mutagenic agents should always be usedsparingly to avoid the introduction of additional extraneous mutationsthat might subsequently complicate the genetic and physiologicalstudies. N-methyl-N'-nitro-n-nitrosoguanidine was used because of itsproven ability to induce temperature-sensitive mutations in culturedmammalian cells (Naha, P. M., "Temperature sensitive conditional mutantsof monkey kidney cells", Nature (London) 223, pp. 1380-1381 (1969);Thompson, L. H., Mankovitz, R., Baker, R. M., Till, J. E. Siminovitch,L., and Whitmore, G. F., "Isolation of temperature-sensitive mutants ofL-cells", Proceedings of the National Academy of Sciences U.S.A. 66, p.377 (1970); Miess, H., and Basilico, C., "Temperature sensitive mutantsof BHK-21 cells", Nature (New Biology) 241, pp. 13-14 (1972)). Under theconditions described in the Materials and Methods section, this potentmutagen did not have a markedly lethal effect on T. gondii. Once theN-methyl-N'-nitro-N-nitrosoguanidine was removed, the treated parasitesgrew as fast as those of the control culture and the yield was reducedonly to one-fourth of the control. This relatively high rate of survivaland brisk growth suggests that the chemical mutagenesis was relativelymild. At least 48 hr. of growth was allowed after the removal of themutagen to allow phenotypic expression of any induced mutations. Duringthis period of growth, the preexisting functional proteins would bediluted and replaced with newly synthesized defective proteins in thoseparasites containing an induced temperature-sensitive mutation.

Selection of Temperature-Sensitive Mutants

Mutagenized T. gondii were cloned by plaque formation at 33° C. inmicrowells as described in the Materials and Methods section. After 2weeks' incubation the wells that contained single plaques wereidentified by low power microscopy. The cells of these singly infectedwells were scraped from the plastic with sterile toothpicks and aliquotsof the resulting suspensions were seeded into duplicate microwell traysalready containing fresh human fibroblast cells. The duplicate cultureswere incubated at 40° or 33° C. for 1 and 2 weeks, respectively, andthen examined for growth of the added parasites. Toxoplasma gondii fromthe vast majority of the clones grew perfectly well at both temperaturesand were discarded as wild types. However, as recorded in Table II,parasites from 1.4% of the mutagenized clones failed to grow at 40° C.although they destroyed the parallel infected cultures at 33° C. Thepheno-type of these possible temperature-sensitive mutants was checkedby repeating the infections at both temperatures, using parasites fromthe well incubated at 33° C. as the inoculum. The confirmed mutants werethen recloned at 33° C. and massively infected cultures were stored inliquid nitrogen. The mutants are numbered serially from "ts-1" to"ts-7".

Many clones of unmutagenized wild-type T. gondii were also examined forthe presence of spontaneous temperature-sensitive mutants. Table IIrecords the negative results of this search. The apparent increasedfrequency of temperature-sensitive mutants after chemical mutagenesis issignificant at the P<0.01 level, suggesting thatN-methyl-N'-nitro-N-nitrosoguanidine is mutagenic for T. gondii.

                  TABLE II                                                        ______________________________________                                        Frequency of Temperature-Sensitive Mutants of Toxoplasma                      gondii in Mutagenized and Unmutagenized Cultures                                              Number of Number of                                           Preliminary treatment                                                                         clones    mutants   Mutants.sup.a                             of T. gondii    tested    isolated  (%)                                       ______________________________________                                        N--methyl-N'--nitro-N--                                                                       486       7         1.4                                       nitrosognanidine.sup.b                                                        None            541       0         0                                         ______________________________________                                         .sup.a The difference between the mutagenized and the unmutagenized clone     is significant at the P < 0.01 level using the chisquared test and one        degree of freedom.                                                            .sup.b The treatment is described in the Materials and Methods section.  

In accordance with the invention, it has now been discovered that aspecific mutant developed as described above (i.e., "ts-4") hasparticular utility for purposes of immunization of mammals withoutattendant chronic infection. A series of tests demonstrating theefficacy of the "ts-4" mutant in this context is presented below.

Inoculation Tests and Results

A variety of tests were performed on laboratory rabbits, squirrelmonkeys and hamsters to determine the immunological activity andpersistence of vaccines including the "ts-4" mutant described above. Incertain additional tests, other infectious strains or antigens (i.e.,RH, T-1, T-45 and killed Toxoplasma antigen (tAg) in Freund's completeadjuvant) were employed. The "ts-4" mutant was propagated in humanforeskin fibroblasts in medium RPMI-1640 with 10% heat inactivated fetalcalf serum and 100 μ/mcg/ml penicillin-streptomycin at 35°-37° C. in anatmosphere of 5% CO₂ in air, until inoculated. The remaining strainswere maintained by serial passage of tachyzoites in mice.

Relevant additional details with respect to the tests are set forthbelow:

TEST 1

In this test laboratory rabbits were inoculated subcutaneously withabout 10⁴ "ts-4" tachyzoites in 9% saline solution, and were challengedafter two and ten months with the RH strain (which is the "parent"strain of the "ts-4" mutant). The results of this test are set forth inTable III. This test demonstrates that the "ts-4"-containing vaccine iseffective for immunization of rabbits. The data also demonstrates thatbetter survival of the rabbits is obtained if they are vaccinated in theear, as opposed to flank vaccination. It is believed that these resultswere obtained because of the temperature-sensitive nature of the "ts-4"mutant, and the fact that the rabbits' ears are somewhat cooler thantheir main bodies. Thus, it is postulated that inoculations in ears orother lower temperature body appendages may give better immunity.

                  TABLE III                                                       ______________________________________                                        Tests for Persistence of Immunity in Rabbits                                               "ts-4"                                                                        Vaccinated.sup.b                                                 Challenge.sup.a                                                                              Flank   Ear        Controls                                    2 months                                                                      10.sup.7 tachyzoites                                                                         s.sup.c s          10.sup.d, 10                                10.sup.5 tachyzoites                                                                         s       s          12, s                                       10.sup.3 tachyzoites                                                                         s       s          12, 14                                      10 months                                                                     10.sup.7 tachyzoites                                                                         8       s, s       12, 18                                      10.sup.5 tachyzoites                                                                         9       s          11, 14                                      10.sup.3 tachyzoites                                                                         --      s          10, 14                                      ______________________________________                                         .sup.a Challenges employed RH strain of T. gondii injected into flanks        .sup.b Subcutaneous (sc) flank injections comprising 10.sup.4 ts4             tachyzoites in 9% saline solution.                                            .sup.c s = animal survived                                                    .sup.d Day of animal death (after challenge)                             

TEST 2 In this test squirrel monkeys were challenged at forty-one andfifty-six days after "ts-4" inoculation. The results of this test areset forth in Table IV. It will be noted that better survival is obtainedwhere two inoculations are given, as opposed to only a singleinoculation; however, the effectiveness of the "ts-4" vaccine ingenerating immunity is manifest.

                  TABLE IV                                                        ______________________________________                                        Pathogenic Challenge of Squirrel Monkeys                                                 Day of.sup.2      Fraction                                                                             Unvaccinated                              "ts-4" inoculation.sup.1                                                                 Challenge Route   Survival                                                                             Controls                                  ______________________________________                                        once       41        sc      1/2    0/2                                       twice.sup.3                                                                              56        sc      1/1    --                                        ______________________________________                                         .sup.1 Subcutaneous (sc) flank injections comprising 10.sup.4 ts4             tachyzoites in 9% saline solution.                                            .sup.2 Days after last inoculation, using T1 strain of T. gondii, 10.sup.     tachyzoites, flank injected.                                                  .sup.3 Second injection occurred 27 days after first injection.          

TEST 3

In this test hamsters were injected with various infectious strains ofT. gondii, in order to determine the pathogenicity thereof. It will benoted in this regard that the "ts-4" strain was completelynon-pathogenic, as opposed to the RH and T-1 strains which resulted in ahigh proportion of hamster deaths (see Table V).

TEST 4

In this test hamsters were again vaccinated with the various Toxoplasmastrains, and weight gains or losses were recorded. The purpose of thistest was to determine whether the "ts-4" vaccine gave rise to non-lethalillnesses in the hamsters. As the results of Table VI demonstrate, theweight gains recorded with respect to the "ts-4" inoculated hamsters arevery close to those of the control hamsters, and it was thereforededuced that the "ts-4" vaccine did not cause debilitating illness inthe hamsters (see Table VI).

TEST 5

In this test hamsters were inoculated with "ts-4" vaccine, and afterfour weeks the hamsters were sacrificed. Various organs of the hamsterswere then ground and put into saline solution and subinoculated ip intomice. Dye test titers of the mice demonstrated that the antibody countwas essentially zero, this occurring four weeks after thesubinoculation. The mice were then challenged with the virulent RH T.gondii strain, with the result that none of the mice survived. The lackof antibody development and of immunity in the recipient micedemonstrates that the "ts-4" vaccination of hamsters does not lead tochronic infection of the hamster organs (see Table VII).

TEST 6

ln this test hamsters vaccinated with "ts-4" mutant were challengedafter various periods of time with the virulent RH and T-1 T. gondiistrains. It will be observed (see Table VIII) that the survival rates ofthe vaccinated hamsters, as opposed to the controls, was very high. Thevaccination of the hamsters occurred only once, and it is believed thatcomplete immunity for all the subjects could be obtained by a doubleinoculation.

TEST 7

In this test respective groups of hamsters were vaccinated with RH,T-45, T-1, ts-4, and tAg in FCA. The first three vaccinations requiredchemoprophylaxis with sulfadiazine. This involved adding sulfadiazine tothe hamsters' drinking water (30-60 mg/100 ml) for three to four weeksafter inoculation, in order to permit the survival of these subjects.The "ts-4" vaccine did not require chemoprophylaxis, nor did thosehamsters inoculated with tAg in FCA. As noted in Table IX, a highpercentage of the hamsters inoculated with live vaccine (i.e, all butthose inoculated with tAg in FCA) survived the RH strain challenge. Inthe case of "ts-4", it is believed that 100% survival can be obtainedusing two separate, spaced in time vaccination doses.

                                      TABLE V                                     __________________________________________________________________________    Survival Fraction and Mean Day of Death of Hamsters                           (60-70 g) Infected with Different Toxoplasma Strains                          Infection      Injected number of tachyzoites                                 Strain                                                                             3.5 × 10.sup.0                                                                 3.5 × 10.sup.1                                                                3.5 × 10.sup.2                                                               3.5 × 10.sup.3                                                               3.5 × 10.sup.4                                                               3.5 × 10.sup.5                                                               3.5 × 10.sup.6                    __________________________________________________________________________    RH   1/5.sup.a,b (12.0).sup.c                                                             0/5 (11.2)                                                                          0/5 (10.6)                                                                         0/6 (9.5)                                                                          0/4 (8.3)                                                                          ND   ND                                      T-1  1/5.sup.a (16.3)                                                                     1/5.sup.a (16.8)                                                                    0/5 (13.8)                                                                         0/5 (15.4)                                                                         0/5 (15.8)                                                                         0/5 (14.6)                                                                         0/5 (13.3)                              T-45 ND.sup.d                                                                             ND    ND   5/5  5/5  5/5  5/5                                     ts-4 5/5.sup.c                                                                            4/4   4/4  6/6  6/6  6/6  3/3                                     __________________________________________________________________________     .sup.a Survivor dye test for antibodies negative.                             .sup.b Number survivors/number in group.                                      .sup.c 3/5 survivors dye test for antibodies negative.                        .sup.d ND = not done.                                                         .sup.6 Mean day of hamster deaths.                                       

                                      TABLE VI                                    __________________________________________________________________________    Effect of Toxoplasma Infection or Vaccination on Body Weight of Hamsters                Week                                                                Toxoplasma strain                                                                       n.sup.a                                                                         O.sup.b                                                                              1    2    3    4                                           __________________________________________________________________________    RH        8 69.0 ± 4.7                                                                        -2.2%.sup.c                                                                        --   --   --                                          T-1       5 129.5 ± 10.1                                                                      ND.sup.d                                                                           -13.3%                                                                             -31.5%                                                                             --                                                    6 148.3 ± 7.4                                                                       ND   -45.4                                                                              --   --                                                    4 114.2 ± 9.6                                                                       ND   -36.8                                                                              --   --                                          T-45      7 116.6 ± 6.9                                                                       -5.8 -10.1                                                                              ND    +1.5                                       ts-4      5 81.4 ± 3.8                                                                        +10.1                                                                              +22.0                                                                              ND   +42.0                                       (tAg-FCA).sup.e                                                                         10                                                                              69.8 ± 3.3                                                                        ND   ND   ND   +44.7                                                 6 69.0 ± 2.6                                                                        ND   ND   ND   +40.4                                       Saline Control                                                                          5 71.2 ± 5.6                                                                        +11.5                                                                              +25.5                                                                              ND   +46.6                                       __________________________________________________________________________     .sup.a Number of hamsters per group.                                          .sup.b Mean group weight in grams the day of challenge or vaccination.        .sup.c Percent change from weight on day of vaccination.                      .sup.d ND = not done.                                                         .sup.e tAgFCA =nonviable Toxoplasma antigen in Freund's complete adjuvant

                  TABLE VII                                                       ______________________________________                                        Subinoculation of Tissues from ts-4 Vaccinated Hamsters                       into Mice to Determine the Presence of Persistent Organisms                   in Hamsters                                                                                 Donors                                                                              Recipients                                                                No. of          Dye  RH                                                       Ham-    No. of  test Challenge                                Subinoculation material                                                                       sters   Mice    Titer                                                                              of Mice                                  ______________________________________                                        ts-4 vaccinated hamsters:.sup.a                                               Lung            8       8       <2   7.9.sup.c                                                                          0/8.sup.d                           Liver           8       8       <2   8.0  0/8                                 Spleen          8       8       <2   7.6  0/8                                 Kidney          8       8       <2   7.9  0/8                                 Adrenal         8       8       <2   7.6  0/8                                 Heart           8       8       <2   8.0  0/8                                 Pancreas        8       8       <2   7.7  0/8                                 Brain           8       8       <2   8.4  0/8                                 Uterus          8       8       <2   8.1  0/8                                 Skeletal muscle & injection                                                                   8       8       <2   8.2  0/8                                 site for ts-4                                                                 Controls for subinoculation                                                   Normal hamster brain                                                                          4       4       <2   7.3  0/4                                 ts-4 tachyzoites (n = 10.sup.0).sup.b                                                         0       4        64  8.0  1/4                                 ts-4 tachyzoites (n = 10.sup.3).sup.b                                                         0       4       1,000                                                                              7.0  3/4                                 ______________________________________                                         .sup.a Hamsters vaccinated with 10.sup.4 ts4, sc, 4 weeks previously.         .sup.b ts4 injected into mice sc.                                             .sup.c Mean day of death, sc challenge was 2 weeks after subinoculation o     tissue.                                                                       .sup.d Number of survivors/number in group.                              

                                      TABLE VIII                                  __________________________________________________________________________    Toxoplasma Challenge of ts-4 Vaccinated Hamsters                              Challenge  Challenge dose and time of challenge after vaccination             Vaccine                                                                            Strain                                                                              2 wk - 10.sup.3                                                                       4 wk - 10.sup.3                                                                       8 wk - 10.sup.3                                                                       16 wk - 10.sup.3                                                                      24 wk - 10.sup.3                   __________________________________________________________________________    ts-4 RH    6/6.sup.a                                                                             6/6     4/5 (10.0)                                                                            5/5     3/5 (13.5 ± .7)                 None RH    0/7 (9.8 ± .4).sup.b                                                               0/6 (9.5 ± 1.2)                                                                    0/5 (10.0 ± 1.2)                                                                   0/4 (9.7 ± .5)                                                                     0/5 (9.2 ± .4)                  ts-4 T-1   ND      ND      ND      6/6     ND                                 None T-1   ND      ND      ND      0/2 (15.0 ± 0)                                                                     ND                                 __________________________________________________________________________     .sup.a Number of survivors/number challenged.                                 .sup.b Mean day of death ± 1SD.                                       

                  TABLE IX                                                        ______________________________________                                        Percent Survival of Hamsters Following RH Strain Challenge                                                       Percent                                                        Time of Challenge                                                                            Survival                                          Type of infection                                                                          after infection                                                                              (5-6                                       Vaccine                                                                              or vaccine   or vaccination per group)                                 ______________________________________                                        RH.sup.a                                                                             Persistent   2, 4, 6, 16, or 24 wk                                                                        100%                                       T-45   Persistent   4, 16, or 24 wk                                                                              100%                                       T-1.sup.a,b                                                                          Persistent   4 or 24 wk     100%                                       ts-4   Non persistent                                                                             2, 4, 16 wk    100%                                                           8 and 24 wk     70%                                       .sup.d tAg in                                                                 Freund's                                                                             killed vaccine                                                                             4 wk            0%                                        complete                                                                      adjuvant                                                                      None   None         2, 4, 8, 16, 24.sup.c                                                                         0%                                        ______________________________________                                         .sup.a Sulfadiazine (90 mg %) chemotherapy was given with the vaccination     .sup.b Survivors of T1 infection.                                             .sup.c Normal control hamsters infected at time vaccinated hamsters were      challenged.                                                                   .sup.d Nonviable Toxoplasma antigen.                                     

TEST 8

This test was designed to determine the amount of time required for theonset of immunity after "ts-4" vaccination of hamsters and mice. ln thistest, the subject animals were inoculated and challenged on the listeddays thereafter with RH T. gondii (see Table X). It will be noted thatthe hamsters achieved virtually complete immunity after four days,whereas in mice the period required for the development of immunity wassomewhat longer.

TEST 9

In this test groups of hamsters were inoculated with the listed strainsof T. gondii or mutants thereof, with the first three groups requiringsulfadiazine chemoprophylaxis for a period of four weeks in order toensure survival. Challenge occurred 112 days after inoculation, byfeeding the hamsters Toxoplasma-infected meat. Inasmuch as ingestion ofinfected meat is the usual means of infection, this test demonstratesthat "ts-4" vaccination provides a very high degree of immunity againstthis usual infective route.

TEST 10

In this test hamsters were inoculated with various doses of "ts-4", andthe buildup of immunity in the animals was thereafter monitored by dyetest titer and RH strain challenge. The challenge occurred 14 days afterinoculation. As the results of Table XII indicate, complete immunityagainst challenge in the hamsters resulted with a dose as small as 20tachyzoites in saline solution.

                  TABLE X                                                         ______________________________________                                        Onset of Immunity to RH Strain Challenge Following                            4 Vaccination                                                                                     Percent                                                                       Survival                                                  Days of   Mean Days of Death                                                                      (5-6 per group)                                           Chal- Ham-              Ham-        Causes of Death.sup.a                     lenge sters   Mice      sters Mice  Days of Death                             ______________________________________                                        0.sup.b                                                                              9.5  .5                                                                              8.6 ± 0.5                                                                             0    0     Pneumonia (9, 10)                         0     10.0 1.2                                                                              8.8 ± 0.8                                                                             0    0     Pneumonia (8,                                                                 10, 11)                                   2     12.5 3.8                                                                              8.8 ± 0.9                                                                             20   0     Pneumonia (10,                                                                12); -     Encephalities (18)             3     23.5 7.5                                                                              ND         60   --    Encephalities                                                                 (18, 29)                                  4     --      7.0 ± 0.7                                                                            100   0       --                                      5     --      ND        100   --      --                                      6     --      10.2 ± 2.8                                                                           100   0       --                                      8     13.0    12.2 ± 2.2                                                                            83   20    Pneumonia (13)                            10    --      11.7 ± 0.6                                                                           100   60      --                                      14    --       8.0      100   .sup. 75.sup.c                                                                        --                                      21    --      19.0      100   83      --                                      ______________________________________                                         .sup.a For hamsters only; the cause of pneumonia or encephalitis was          Toxoplasma.                                                                   .sup.b No vaccine.                                                            .sup.c 4 mice in this group.                                             

                  TABLE XI                                                        ______________________________________                                        Immunity to Oral Challenge with T-1 Toxoplasma                                Bradyzoites = meat                                                                    Number of Deaths                                                              per         Mean Day  challenged                                              Number per Group                                                                          of Death  of survivors.sup.b                              ______________________________________                                        Hamsters                                                                      RH chronic                                                                               0/12         --        all survive                                 T-1 chronic                                                                             0/5           --        "                                           T-45 chronic                                                                            0/4           --        "                                           ts-4 vaccinated                                                                          0/12         --        "                                           Normal    6/9           7.5 ± 1.4.sup.a                                                                      died                                        Mice                                                                          Normal    5/5           8.0 ± 1.9.sup.                                                                       died                                        ______________________________________                                         .sup.a Three survivors dye tests titers >1,000, 2 weeks.                      .sup.b 10.sup.5 RH tachyzoites, administered sc.                         

                  TABLE XII                                                       ______________________________________                                        The Effect of Different Doses of ts-4 on the Estabilishment of                Protective Immunity and Antibody Response 2 weeks After                       Vaccination of Hamsters and Mice                                              Counted dose         Hamsters                                                 of ts-4      Dye test titer.sup.a                                                                      RH challenge.sup.b                                   ______________________________________                                        2 × 10.sup.4                                                                         64,000      4/4                                                    × 10.sup.3                                                                         16,000      4/4                                                    × 10.sup.2                                                                         16,000      4/4                                                    × 10.sup.1                                                                         32,000      4/4                                                               8,000                                                                         4,000                                                                         4,000                                                             × 10.sup.0                                                                          16,000      .sup. (9.3).sup.c                                                 4,000                                                                         <4                                                                            <4                                                                            <4                                                                × 10.sup.-1                                                                         <2          0/4 (9.2)                                            None         <2          0/5 (9.2)                                            ______________________________________                                         .sup.a Dye test titers result of test on pool of .1 ml serum from each        animal in group. Titers from hamsters given 10.sup.0, 10.sup.1 were           individually determined.                                                      .sup.b Number of survivors/number challenged with 10.sup.3 RH strain,         administered sc two weeks after vaccination.                                  .sup.c Mean day of death.                                                

We claim:
 1. A method of immunizing man, cattle, sheep, goats, birds onzoo mammals against Toxoplasma gondii, comprising the step ofadministering thereto by subcutaneous injection an amount of a mutant ofToxoplasma gondii for developing immunity to Toxoplasma gondii challengewithout concomitant chronic Toxoplasma gondii infection, said mutantbeing designated ts-4 by the American Type Culture Collection and havingaccession number
 40050. 2. The method of claim 1, wherein said mutant isinjected at a level of at least about 20 tachyzoites.
 3. The method ofclaim 1, wherein man is immunized.